- Email: [email protected]
Comparison of l -serine uptake by human placental microvillous membrane vesicles and placental villous fragments
Placenta 31 (2010) 456–459
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Short Communication
Comparison of L-serine uptake by human placental microvillous membrane vesicles and placental villous fragments A.P. Brand a, S.L. Greenwood b, J.D. Glazier b, E.J. Bennett a, K.M. Godfrey a, C.P. Sibley b, M.A. Hanson a, R.M. Lewis a, * a
University of Southampton, School of Medicine, UK Maternal and Fetal Health Research Group, School of Clinical and Laboratory Sciences, Manchester Academic Health Science Centre, University of Manchester, St. Mary’s Hospital, Manchester, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 28 January 2010
Both syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments are used to characterize the placental uptake of maternal substrate and to investigate changes in uptake associated with pathological conditions. However, the two techniques have not been directly compared. In this study uptake of 14C-L-serine was compared in placental villous fragments and in MVM prepared from the same placentas. 14 C-L-serine uptake into MVM vesicles was mediated by System L and System A and smaller unidentified Naþ-dependent and Naþ-independent components. In villous fragments an unidentified Naþ-dependent component mediated the majority of 14C-L-serine uptake followed by System A and System L. The unidentified Naþ-independent component of L-serine uptake was not detected in villous fragments. The ratio of System A activity to System L activity was similar in villous fragments and MVM vesicles. However, the unidentified Naþ-dependent component in villous fragments was significantly higher than that in MVM vesicles. This indicates that the main differences in serine uptake mechanisms identified using the two techniques were not due to differences in System A and System L activity but to differences in the unidentified Naþ-dependent component. This study suggests that uptake of L-serine into MVM vesicles and villous fragments via Systems A and L is comparable, but that this is not true for all components of L-serine uptake. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Amino acid Transport System A System L
1. Introduction Both microvillous plasma membrane (MVM) vesicles [1] and placental villous fragments [2] are used to study uptake of substrates, particularly amino acids, by MVM of the human placental syncytiotrophoblast. These techniques have been used to characterize transport systems and to determine whether they are altered in pathological situations [3–7]. However, to date there have been no studies directly comparing transport data obtained by these two preparations from the same placenta. The placental villous fragments technique measures the accumulation of radiolabelled solutes into small pieces (1–2 mm3) of intact placental villi collected soon after delivery [2]. In this model there is preservation of cellular architecture and regulatory
* Corresponding author. Institute of Developmental Sciences, University of Southampton, MP 887 Southampton General Hospital, Tremona Road, SO16 6YD, UK. Tel.: þ44 2380798663. E-mail address: [email protected] (R.M. Lewis). 0143-4004/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.01.016
mechanisms of the intact cell. As the villi are surrounded by syncytiotrophoblast, nutrient uptake by the villous fragments will predominantly represent uptake across the MVM. However, the villous fragments have a large extracellular space and contain several different cell types which, although surrounded by syncytiotrophoblast, could also contribute to uptake. MVM vesicles can be prepared by a number of methods [8,9]. These techniques use the activity of alkaline phosphatase, an enzyme predominantly expressed on the MVM, as a marker of enrichment [10]. MVM vesicles have the advantage that uptake of solute in this model is across a defined syncytiotrophoblast plasma membrane, with the same orientation as in vivo [1,8]. However transport into vesicles will lack normal intracellular regulation, as they are devoid of intracellular components. Placental uptake of amino acids from maternal plasma is mediated by transporters on the MVM of the syncytiotrophoblast [11]. We have previously demonstrated that L-serine uptake into MVM vesicles is mediated by System A and System L as well as smaller unidentified Naþ-dependent and Naþ-independent components [4].
A.P. Brand et al. / Placenta 31 (2010) 456–459
These techniques are important models to investigate the effects of pathological conditions on placental nutrient transport [6,7]. However, to date there has been no direct comparison of amino acid uptake using these two approaches to determine whether there is comparability in their transport characteristics. To address this issue we have characterised uptake of 14C-L-serine, a substrate which is transported by both Naþ-dependent and Naþ-independent mechanisms, into placental villous fragments and in MVM vesicles prepared from the same placentas. 2. Methods Human tissues were collected after informed written consent and with the approval of the South and West Hampshire Local Research Ethics Committee. Placentas were collected from term uncomplicated pregnancies following vaginal or caesarean section within 30 min of delivery. 2.1. Reagents 14 C-L-serine was supplied by Perkin Elmer Life Sciences, Boston, MA, USA. Amino acids and amino acid analogues were supplied by Sigma Aldrich, Poole, Dorset, UK.
2.2. Placental villous fragment uptake Placental villous fragments were dissected from freshly delivered placentas in Tyrode’s buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose adjusted to pH 7.4) at room temperature (z20 C) and suspended on cotton threads in Tyrode’s buffer at 37 C. Placental villous fragments were preincubated for 30 min in Tyrode’s buffer at 37 C then incubated for 2 min in either Tyrode’s or Naþ-free Tyrode’s (with sodium chloride replaced by equimolar choline chloride) at 37 C before being transferred to Tyrode’s or Naþ-free Tyrode’s containing 3 mmol/L L-[14C(U)]-serine and any inhibitors. Villous fragments were then incubated for 15 min at 37 C (uptake of 3 mM L-[14C(U)]-serine was confirmed to be linear over 5–25 min) and then washed twice for 15 s in ice-cold Tyrode’s or Naþ-free Tyrode’s. Villous fragments were incubated for 18–24 h in 4 mL water to induce cellular lysis and solubilised overnight in 3 mL 0.3 M NaOH at 37 C. Radiolabel was counted in the lysate in a liquid scintillation counter (LKB Wallac, Turku, Finland). Following neutralisation, fragment protein content in the NaOH lysate was assayed using the Biorad Protein Assay. 2.3. MVM vesicle preparation and uptakes MVM vesicles were purified as described previously [4]. Uptakes were performed under two sets of conditions the standard method as described previously [4], and an adapted method where conditions more closely resembled those in the villous fragment method, allowing direct comparison. Standard method: Uptake of 7.5 mmol/L 14C-L-serine were performed at room temperature (z20 C) and initiated by combining 20 mL of MVM vesicle mixture (z7.5 mg protein/ml) in intra-vesicular buffer (290 mM sucrose, 5 mM tris, 5 mM Hepes, pH 7.4) with 20 mL of extra-vesicular buffer (EVB; 5 mM Hepes, 5 mM tris and either 145 mM NaCl or KCl, pH 7.4) containing tracer and any inhibitors. Uptake was stopped at 15 s by the addition of 2 mL ice-cold Krebs ringer phosphate buffer (130 mM NaCl, 10 mM Na2HPO4, 4.2 mM KCl, 1.2 mM MgSO4, 0.75 mM CaCl2, pH 7.4) and filtered through a HAWP nitrocellulose filter (Millipore, 0.45 mm). The radioactivity on the filters was determined by liquid scintillation counting. Adapted MVM vesicle uptakes: To investigate whether differences in buffer composition or 14C-serine concentration had any effect on L-serine uptake, the MVM vesicle studies were repeated in 5 of the 6 placentas using an adapted protocol. EVB was made with 145 mM choline chloride in place of KCl and with 3 mM L-[14C(U)]serine, as used in the fragment studies. Experiments were also performed to validate the MVM vesicle studies at 37 C, but it was not possible to demonstrate linear uptake of 14C-L-serine under these conditions. 2.4. Identification of different uptake components using competitive inhibition Uptakes were performed in the presence of competitive inhibitors and the presence or absence of Naþ. L-Serine uptake via System A was taken to be Naþ-dependent uptake that was inhibitable by a-methylaminoisobutyric acid (MeAIB). L-Serine uptake mediated by System L was taken to be the Naþ-independent L-serine uptake inhibitable by 2-norbornanecarboxylic acid (BCH). Non-specific uptake was taken to be the component of 14C-serine uptake that was not inhibitable with cold serine. For MVM vesicle studies, inhibitors were used at 5 mM as this concentration completely inhibits serine uptake [4]. For placental villous fragments studies, inhibitors were used at 11 mM based on our previous demonstration that 10 mM MeAIB completely inhibits Naþ-dependent 14C-MeAIB uptake (data not shown).
457
2.5. Statistics Differences between uptake components were analysed using the SPSS version 17 analysis package (SPSS Inc, Chicago, Il, USA) with a one-way ANOVA followed by Bonferonni post hoc test where appropriate. Significance was assumed where P < 0.05. Data is presented as mean SEM.
3. Results Fragments: Uptake of 3 mM 14C-L-serine into placental villous fragments occurred at a rate of 70.9 3.3 pmol/mg protein/15 min and was mediated by Naþ-dependent and Naþ-independent mechanisms. Naþ-dependent uptake accounted for 77.4 3.2% of total L-serine uptake of which 13.2 3.1% was shown to be mediated by System A (MeAIB-inhibitable) and 64.2 3.2% by non System A-mediated mechanisms. Naþ-independent uptake (22.0 3.7% of total L-serine uptake) was all accounted for by System L (BCH-inhibitable) which accounted for 23.3 4.9% of total L-serine uptake (n ¼ 6, Fig. 1). Vesicles: Two protocols were performed; the first under standard conditions as described previously (Lewis et al., 2007) and the second using the same concentration of tracer as in the fragment experiments, and replacement of KCl by choline chloride when measuring Naþ-independent uptake. Uptake of 7.5 mM 14C-L-serine into MVM vesicles occurred at a rate of 15.4 2.7 pmol/mg protein/ min while uptake of 3 mM 14C-L-serine into MVM vesicles occurred at a rate of 18.9 5.5 pmol/mg protein/min. The uptake rates in the two conditions were not significantly different. Using both the standard and adapted protocols, L-serine uptake was mediated by Naþ-dependent and Naþ-independent mechanisms (Fig. 1). Comparing the standard (n ¼ 6) and adapted (n ¼ 5) MVM vesicle protocols, there were no significant differences in the proportion of L-serine uptake by Naþ-dependent and Naþ-independent mechanisms nor in the proportion of L-serine uptake attributable to System A (MeAIB-inhibitable) or System L (BCHinhibitable, Naþ-independent uptake) (Fig. 1). The ratio of Naþ-dependent to Naþ-independent activity (14C-Lserine uptake in pmol/mg protein/unit time) in MVM vesicles was significantly different to that seen in villous fragments from the same placentas (Fig. 2). The ratio of System A to System L activity in MVM vesicles was not significantly different to that in villous fragments (Fig. 2). The ratio of System L to Naþ-independent activity in MVM vesicles was significantly decreased compared to that in placental villous fragments (P < 0.05, Fig. 2). The ratio of System A to unidentified Naþ-dependent activity in MVM vesicles was significantly greater than in villous fragments (P < 0.05, Fig. 2). 4. Discussion This study demonstrates that in MVM vesicles and villous fragments prepared from the same placentas there were no significant differences in the relative uptake of L-serine by Systems A and L but significant differences in other uptake components were apparent. These observations suggest that specific transport components exhibit differential activity between the two techniques. This study increases our confidence that these techniques accurately represent the physiological activity of systems A and L. In contrast, the activity of the unidentified Naþ-dependent component was higher in villous fragments than in MVM vesicles. This demonstrates that for specific transport systems there are differences between the techniques and it is unclear which technique best represents physiological activity of these components. The main difference between the techniques was serine uptake by the unidentified Naþ-dependent component. Two questions arise with regard to this component; why is it so much greater in the fragments than the vesicles and what is the transporter which
458
A.P. Brand et al. / Placenta 31 (2010) 456–459
1
Placental villous fragments
Proportional serine uptake
MVM vesicles standard protocol MVM vesicles adapted protocol
0.75
φ φ
φ φ
* *
φ φ
φ φ
* *
0.5
0.25
0 Na+ dependent
Na+ independent
System A
Unknown Na+ dependent
System L
Unknown Na+ independent
Transport component Fig. 1. Comparison of 14C-L-serine uptake into placental villous fragments and MVM vesicles prepared from the same placentas. The MVM vesicle uptakes were conducted using our standard protocol (n ¼ 6) and with an adapted protocol (n ¼ 5) where the tracer concentration was reduced from 7.5 to 3 mM as used the fragment studies and of Naþ independent uptake was measured in the presence of choline chloride, rather than KCl, as in the fragment studies. There are significant differences in the proportion of uptake by different components, primarily due to a difference in the Naþ-dependent uptake which was not mediated by system A. Significant differences between the MVM vesicles and villous fragments are indicated by *P < 0.05 vs villous fragments, fP < 0.001 vs fragments. Data is mean SEM.
mediates it? The reason for the apparent difference in the unidentified proportion of Naþ-dependent serine uptake component between vesicles and fragments is not clear although there are several possible explanations. The villous fragments contain other cell types which, although surrounded by syncytiotrophoblast, could contribute to uptake, particularly at the cut ends. In MVM vesicles transport proteins may be damaged during the purification process or lose an intracellular cofactor required for their function. Any transporters stored in vesicles within the cytoplasm would also be lost in the MVM vesicle preparations whereas these could be recruited to the MVM in villous fragments [12–14]. Other explanations may include differences in the Naþ gradient in the two systems or due to the gradient of another co-transported ion (e.g. Cl, Kþ or Hþ) which could selectively affect particular transport systems. Trans-stimulation by intracellular amino acids in the villous fragments might have been expected to cause a relative increase in System L activity in villous fragments, but this did not appear to be the case. As villous fragments are metabolically active, the oxidation of serine may have resulted in loss of tracer as CO2. While this could mean that the magnitude of serine uptake is underestimated, the proportions of uptake by different systems will remain the same. If
the unidentified Naþ-dependent component were temperature sensitive, this could also contribute to the observed difference between the two techniques. We attempted to address this issue by measuring serine uptake into MVM vesicles at 37 C. However, under these conditions serine uptake into MVM vesicles was not linear with time and we were not able to resolve the issue. Transporters known to mediate Naþ-dependent serine uptake in placental tissue include the System A isoforms SNAT1, 2 and 4 (SLC38A1, 2 and 4) and ASC (SCL1A4 and 5) [11]. Naþ-dependent serine transporters not known to be active in placenta include the System B0 isoform B0AT1 (SCL6A19), System B0,þ (SLC6A15) and the System N isoform SNAT5 (SLC38A5) based on functional studies in MVM and BM vesicles. However, the most likely candidates for this unidentified Naþ-dependent system, based on substrate specificity and known localisation to the syncytiotrophoblast, would be isoforms of System A and System ASC. The System A isoforms SNAT1 and 2 should be completely inhibited by the concentrations of MeAIB used in these studies. However it is possible that SNAT4, which has a lower affinity for MeAIB is not fully inhibited by the concentration of MeAIB used in the fragment studies [15]. Although SNAT4 activity is demonstrable
5
proportional activit y
Placental villous fragments MVM vesicles standard protocol MVM vesicles adapted protocol
4
3
2
φ φ
φ
φ
φ
φ
*
*
1
0 Na+ dependent / Na+ independent
System A / System L
Unknown Na+ dependent / Na+ independent
System A / Na+ dependent
System L / Na+ independent
Fig. 2. Relative serine uptake by different transport components in placental villous fragments and MVM vesicles measured in the same placentas. While the relative activity of system A and system L were not significantly different there were differences between the techniques in relation to Naþ dependent/Naþ independent activity and system A/Naþ dependent activity. This indicates that while there are differences between the methodologies for some transport components others, such as system A and system L, are comparable. *P < 0.05 vs villous fragments, fP < 0.001 vs villous fragments. Data is mean SEM, n ¼ 5 or 6.
A.P. Brand et al. / Placenta 31 (2010) 456–459
in first trimester placental villous fragments and MVM vesicles isolated from term placentas, there is little or no detectable SNAT4 activity in term placental villous fragments [16]. As such we think it is unlikely that this component of serine uptake is SNAT4mediated. System ASC activity has been demonstrated in the BM but not MVM of placental syncytiotrophoblast [17,18]. If the unidentified Naþ-dependent component of serine uptake was by System ASC, our study suggests it has low activity in vesicles and there may not have been enough activity for definitive characterisation. Without further experimentation it is not clear which transport system is mediating the unidentified Naþ-dependent serine uptake. While SNAT4 or System ASC would be the most obvious candidates, other transport systems cannot be ruled out. These techniques are important for the study of amino aid transporters in fetal growth restriction. Most work in this area has focused on systems A and L which were found to be comparable between the two techniques. However this work does raise the question as to how well these techniques represent less well described transport components, which might be important in fetal growth restriction. In conclusion, we have shown that key components of serine uptake are comparable between techniques but that for specific components of L-serine uptake, such as Systems A and L, there are significant differences between the MVM vesicle and placental villous fragment techniques. This provides reassurance that both models can be exploited to examine changes in placental transporter activity in well-characterised transporters. This notion is further supported by the similar trend observed between MVM vesicles and placental villous fragments with regard to the gestational increase in System A activity [16,19] and the reduced activity of Systems A [20,21] and L [22,23] associated with fetal growth restriction.
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17] [18] [19]
References [1] Glazier JD, Sibley CP. In vitro methods for studying human placental amino acid transport: placental plasma membrane vesicles. Methods Mol Med 2006;122:241–52. [2] Greenwood SL, Sibley CP. In vitro methods for studying human placental amino acid transport placental villous fragments. Methods Mol Med 2006;122:253–64. [3] Ayuk PT, Sibley CP, Donnai P, D’Souza S, Glazier JD. Development and polarization of cationic amino acid transporters and regulators in the human placenta. Am J Physiol Cell Physiol 2000;278:C1162–71. [4] Lewis RM, Glazier J, Greenwood SL, Bennett EJ, Godfrey KM, Jackson AA, et al. L-serine uptake by human placental microvillous membrane vesicles. Placenta 2007;28:445–52. [5] Godfrey KM, Matthews N, Glazier J, Jackson A, Wilman C, Sibley CP. Neutral amino acid uptake by the microvillous plasma membrane of the human
[20]
[21]
[22] [23]
459
placenta is inversely related to fetal size at birth in normal pregnancy. J Clin Endocrinol Metab 1998;83:3320–6. Johansson M, Karlsson L, Wennergren M, Jansson T, Powell TL. Activity and protein expression of Naþ/Kþ ATPase are reduced in microvillous syncytiotrophoblast plasma membranes isolated from pregnancies complicated by intrauterine growth restriction. J Clin Endocrinol Metab 2003;88:2831–7. Magnusson AL, Waterman IJ, Wennergren M, Jansson T, Powell TL. Triglyceride hydrolase activities and expression of fatty acid binding proteins in the human placenta in pregnancies complicated by intrauterine growth restriction and diabetes. J Clin Endocrinol Metab 2004;89:4607–14. Glazier JD, Jones CJ, Sibley CP. Purification and Naþ uptake by human placental microvillus membrane vesicles prepared by three different methods. Biochim Biophys Acta 1988;945:127–34. Illsley NP, Wang ZQ, Gray A, Sellers MC, Jacobs MM. Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta 1990;1029:218–26. Jimenez V, Henriquez M, Llanos P, Riquelme G. Isolation and purification of human placental plasma membranes from normal and pre-eclamptic pregnancies. A comparative study. Placenta 2004;25:422–37. Cleal JK, Lewis RM. The mechanisms and regulation of placental amino acid transport to the human foetus. J Neuroendocrinol 2008;20:419–26. Ling R, Bridges CC, Sugawara M, Leibach FH, Prasad PD, Ganapathy V, et al. Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A. Biochim Biophys Acta 2001;1512:15–21. Hatanaka T, Hatanaka Y, Tsuchida J, Ganapathy V, Setou M. Amino acid transporter ATA2 is stored at the trans-golgi network and released by insulin stimulus in adipocytes. J Biol Chem 2006;281:39273–84. Parrott MS, von Versen-Hoeynck F, Ness RB, Markovic N, Roberts JM. System A amino acid transporter activity in term placenta is substrate specific and inversely related to amino acid concentration. Reprod Sci 2007;14:687–93. Hatanaka T, Huang W, Ling R, Prasad PD, Sugawara M, Leibach FH, et al. Evidence for the transport of neutral as well as cationic amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A. Biochim Biophys Acta 2001;1510:10–7. Desforges M, Mynett KJ, Jones RL, Greenwood SL, Westwood M, Sibley CP, et al. The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane. J Physiol 2009;587:61–72. Hoeltzli SD, Smith CH. Alanine transport systems in isolated basal plasma membrane of human placenta. Am J Physiol 1989;256:C630–7. Johnson LW, Smith CH. Neutral amino acid transport systems of microvillous membrane of human placenta. Am J Physiol 1988;254:C773–80. Mahendran D, Byrne S, Donnai P, D’Souza SW, Glazier JD, Jones CJ, et al. Naþ transport, Hþ concentration gradient dissipation, and system A amino acid transporter activity in purified microvillous plasma membrane isolated from first-trimester human placenta: comparison with the term microvillous membrane. Am J Obstet Gynecol 1994;171:1534–40. Mahendran D, Donnai P, Glazier JD, D’Souza SW, Boyd RD, Sibley CP. Amino acid (system A) transporter activity in microvillous membrane vesicles from the placentas of appropriate and small for gestational age babies. Pediatr Res 1993;34:661–5. Shibata E, Hubel CA, Powers RW, von Versen-Hoeynck F, Gammill H, Rajakumar A, et al. Placental system A amino acid transport is reduced in pregnancies with small for gestational age (SGA) infants but not in preeclampsia with SGA infants. Placenta 2008;29:879–82. Jansson T, Scholtbach V, Powell TL. Placental transport of leucine and lysine is reduced in intrauterine growth restriction. Pediatr Res 1998;44:532–7. Roos S, Jansson N, Palmberg I, Saljo K, Powell TL, Jansson T. Mammalian target of rapamycin in the human placenta regulates leucine transport and is downregulated in restricted fetal growth. J Physiol 2007;582:449–59.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Short Communication
Comparison of L-serine uptake by human placental microvillous membrane vesicles and placental villous fragments A.P. Brand a, S.L. Greenwood b, J.D. Glazier b, E.J. Bennett a, K.M. Godfrey a, C.P. Sibley b, M.A. Hanson a, R.M. Lewis a, * a
University of Southampton, School of Medicine, UK Maternal and Fetal Health Research Group, School of Clinical and Laboratory Sciences, Manchester Academic Health Science Centre, University of Manchester, St. Mary’s Hospital, Manchester, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 28 January 2010
Both syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments are used to characterize the placental uptake of maternal substrate and to investigate changes in uptake associated with pathological conditions. However, the two techniques have not been directly compared. In this study uptake of 14C-L-serine was compared in placental villous fragments and in MVM prepared from the same placentas. 14 C-L-serine uptake into MVM vesicles was mediated by System L and System A and smaller unidentified Naþ-dependent and Naþ-independent components. In villous fragments an unidentified Naþ-dependent component mediated the majority of 14C-L-serine uptake followed by System A and System L. The unidentified Naþ-independent component of L-serine uptake was not detected in villous fragments. The ratio of System A activity to System L activity was similar in villous fragments and MVM vesicles. However, the unidentified Naþ-dependent component in villous fragments was significantly higher than that in MVM vesicles. This indicates that the main differences in serine uptake mechanisms identified using the two techniques were not due to differences in System A and System L activity but to differences in the unidentified Naþ-dependent component. This study suggests that uptake of L-serine into MVM vesicles and villous fragments via Systems A and L is comparable, but that this is not true for all components of L-serine uptake. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Amino acid Transport System A System L
1. Introduction Both microvillous plasma membrane (MVM) vesicles [1] and placental villous fragments [2] are used to study uptake of substrates, particularly amino acids, by MVM of the human placental syncytiotrophoblast. These techniques have been used to characterize transport systems and to determine whether they are altered in pathological situations [3–7]. However, to date there have been no studies directly comparing transport data obtained by these two preparations from the same placenta. The placental villous fragments technique measures the accumulation of radiolabelled solutes into small pieces (1–2 mm3) of intact placental villi collected soon after delivery [2]. In this model there is preservation of cellular architecture and regulatory
* Corresponding author. Institute of Developmental Sciences, University of Southampton, MP 887 Southampton General Hospital, Tremona Road, SO16 6YD, UK. Tel.: þ44 2380798663. E-mail address: [email protected] (R.M. Lewis). 0143-4004/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.01.016
mechanisms of the intact cell. As the villi are surrounded by syncytiotrophoblast, nutrient uptake by the villous fragments will predominantly represent uptake across the MVM. However, the villous fragments have a large extracellular space and contain several different cell types which, although surrounded by syncytiotrophoblast, could also contribute to uptake. MVM vesicles can be prepared by a number of methods [8,9]. These techniques use the activity of alkaline phosphatase, an enzyme predominantly expressed on the MVM, as a marker of enrichment [10]. MVM vesicles have the advantage that uptake of solute in this model is across a defined syncytiotrophoblast plasma membrane, with the same orientation as in vivo [1,8]. However transport into vesicles will lack normal intracellular regulation, as they are devoid of intracellular components. Placental uptake of amino acids from maternal plasma is mediated by transporters on the MVM of the syncytiotrophoblast [11]. We have previously demonstrated that L-serine uptake into MVM vesicles is mediated by System A and System L as well as smaller unidentified Naþ-dependent and Naþ-independent components [4].
A.P. Brand et al. / Placenta 31 (2010) 456–459
These techniques are important models to investigate the effects of pathological conditions on placental nutrient transport [6,7]. However, to date there has been no direct comparison of amino acid uptake using these two approaches to determine whether there is comparability in their transport characteristics. To address this issue we have characterised uptake of 14C-L-serine, a substrate which is transported by both Naþ-dependent and Naþ-independent mechanisms, into placental villous fragments and in MVM vesicles prepared from the same placentas. 2. Methods Human tissues were collected after informed written consent and with the approval of the South and West Hampshire Local Research Ethics Committee. Placentas were collected from term uncomplicated pregnancies following vaginal or caesarean section within 30 min of delivery. 2.1. Reagents 14 C-L-serine was supplied by Perkin Elmer Life Sciences, Boston, MA, USA. Amino acids and amino acid analogues were supplied by Sigma Aldrich, Poole, Dorset, UK.
2.2. Placental villous fragment uptake Placental villous fragments were dissected from freshly delivered placentas in Tyrode’s buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose adjusted to pH 7.4) at room temperature (z20 C) and suspended on cotton threads in Tyrode’s buffer at 37 C. Placental villous fragments were preincubated for 30 min in Tyrode’s buffer at 37 C then incubated for 2 min in either Tyrode’s or Naþ-free Tyrode’s (with sodium chloride replaced by equimolar choline chloride) at 37 C before being transferred to Tyrode’s or Naþ-free Tyrode’s containing 3 mmol/L L-[14C(U)]-serine and any inhibitors. Villous fragments were then incubated for 15 min at 37 C (uptake of 3 mM L-[14C(U)]-serine was confirmed to be linear over 5–25 min) and then washed twice for 15 s in ice-cold Tyrode’s or Naþ-free Tyrode’s. Villous fragments were incubated for 18–24 h in 4 mL water to induce cellular lysis and solubilised overnight in 3 mL 0.3 M NaOH at 37 C. Radiolabel was counted in the lysate in a liquid scintillation counter (LKB Wallac, Turku, Finland). Following neutralisation, fragment protein content in the NaOH lysate was assayed using the Biorad Protein Assay. 2.3. MVM vesicle preparation and uptakes MVM vesicles were purified as described previously [4]. Uptakes were performed under two sets of conditions the standard method as described previously [4], and an adapted method where conditions more closely resembled those in the villous fragment method, allowing direct comparison. Standard method: Uptake of 7.5 mmol/L 14C-L-serine were performed at room temperature (z20 C) and initiated by combining 20 mL of MVM vesicle mixture (z7.5 mg protein/ml) in intra-vesicular buffer (290 mM sucrose, 5 mM tris, 5 mM Hepes, pH 7.4) with 20 mL of extra-vesicular buffer (EVB; 5 mM Hepes, 5 mM tris and either 145 mM NaCl or KCl, pH 7.4) containing tracer and any inhibitors. Uptake was stopped at 15 s by the addition of 2 mL ice-cold Krebs ringer phosphate buffer (130 mM NaCl, 10 mM Na2HPO4, 4.2 mM KCl, 1.2 mM MgSO4, 0.75 mM CaCl2, pH 7.4) and filtered through a HAWP nitrocellulose filter (Millipore, 0.45 mm). The radioactivity on the filters was determined by liquid scintillation counting. Adapted MVM vesicle uptakes: To investigate whether differences in buffer composition or 14C-serine concentration had any effect on L-serine uptake, the MVM vesicle studies were repeated in 5 of the 6 placentas using an adapted protocol. EVB was made with 145 mM choline chloride in place of KCl and with 3 mM L-[14C(U)]serine, as used in the fragment studies. Experiments were also performed to validate the MVM vesicle studies at 37 C, but it was not possible to demonstrate linear uptake of 14C-L-serine under these conditions. 2.4. Identification of different uptake components using competitive inhibition Uptakes were performed in the presence of competitive inhibitors and the presence or absence of Naþ. L-Serine uptake via System A was taken to be Naþ-dependent uptake that was inhibitable by a-methylaminoisobutyric acid (MeAIB). L-Serine uptake mediated by System L was taken to be the Naþ-independent L-serine uptake inhibitable by 2-norbornanecarboxylic acid (BCH). Non-specific uptake was taken to be the component of 14C-serine uptake that was not inhibitable with cold serine. For MVM vesicle studies, inhibitors were used at 5 mM as this concentration completely inhibits serine uptake [4]. For placental villous fragments studies, inhibitors were used at 11 mM based on our previous demonstration that 10 mM MeAIB completely inhibits Naþ-dependent 14C-MeAIB uptake (data not shown).
457
2.5. Statistics Differences between uptake components were analysed using the SPSS version 17 analysis package (SPSS Inc, Chicago, Il, USA) with a one-way ANOVA followed by Bonferonni post hoc test where appropriate. Significance was assumed where P < 0.05. Data is presented as mean SEM.
3. Results Fragments: Uptake of 3 mM 14C-L-serine into placental villous fragments occurred at a rate of 70.9 3.3 pmol/mg protein/15 min and was mediated by Naþ-dependent and Naþ-independent mechanisms. Naþ-dependent uptake accounted for 77.4 3.2% of total L-serine uptake of which 13.2 3.1% was shown to be mediated by System A (MeAIB-inhibitable) and 64.2 3.2% by non System A-mediated mechanisms. Naþ-independent uptake (22.0 3.7% of total L-serine uptake) was all accounted for by System L (BCH-inhibitable) which accounted for 23.3 4.9% of total L-serine uptake (n ¼ 6, Fig. 1). Vesicles: Two protocols were performed; the first under standard conditions as described previously (Lewis et al., 2007) and the second using the same concentration of tracer as in the fragment experiments, and replacement of KCl by choline chloride when measuring Naþ-independent uptake. Uptake of 7.5 mM 14C-L-serine into MVM vesicles occurred at a rate of 15.4 2.7 pmol/mg protein/ min while uptake of 3 mM 14C-L-serine into MVM vesicles occurred at a rate of 18.9 5.5 pmol/mg protein/min. The uptake rates in the two conditions were not significantly different. Using both the standard and adapted protocols, L-serine uptake was mediated by Naþ-dependent and Naþ-independent mechanisms (Fig. 1). Comparing the standard (n ¼ 6) and adapted (n ¼ 5) MVM vesicle protocols, there were no significant differences in the proportion of L-serine uptake by Naþ-dependent and Naþ-independent mechanisms nor in the proportion of L-serine uptake attributable to System A (MeAIB-inhibitable) or System L (BCHinhibitable, Naþ-independent uptake) (Fig. 1). The ratio of Naþ-dependent to Naþ-independent activity (14C-Lserine uptake in pmol/mg protein/unit time) in MVM vesicles was significantly different to that seen in villous fragments from the same placentas (Fig. 2). The ratio of System A to System L activity in MVM vesicles was not significantly different to that in villous fragments (Fig. 2). The ratio of System L to Naþ-independent activity in MVM vesicles was significantly decreased compared to that in placental villous fragments (P < 0.05, Fig. 2). The ratio of System A to unidentified Naþ-dependent activity in MVM vesicles was significantly greater than in villous fragments (P < 0.05, Fig. 2). 4. Discussion This study demonstrates that in MVM vesicles and villous fragments prepared from the same placentas there were no significant differences in the relative uptake of L-serine by Systems A and L but significant differences in other uptake components were apparent. These observations suggest that specific transport components exhibit differential activity between the two techniques. This study increases our confidence that these techniques accurately represent the physiological activity of systems A and L. In contrast, the activity of the unidentified Naþ-dependent component was higher in villous fragments than in MVM vesicles. This demonstrates that for specific transport systems there are differences between the techniques and it is unclear which technique best represents physiological activity of these components. The main difference between the techniques was serine uptake by the unidentified Naþ-dependent component. Two questions arise with regard to this component; why is it so much greater in the fragments than the vesicles and what is the transporter which
458
A.P. Brand et al. / Placenta 31 (2010) 456–459
1
Placental villous fragments
Proportional serine uptake
MVM vesicles standard protocol MVM vesicles adapted protocol
0.75
φ φ
φ φ
* *
φ φ
φ φ
* *
0.5
0.25
0 Na+ dependent
Na+ independent
System A
Unknown Na+ dependent
System L
Unknown Na+ independent
Transport component Fig. 1. Comparison of 14C-L-serine uptake into placental villous fragments and MVM vesicles prepared from the same placentas. The MVM vesicle uptakes were conducted using our standard protocol (n ¼ 6) and with an adapted protocol (n ¼ 5) where the tracer concentration was reduced from 7.5 to 3 mM as used the fragment studies and of Naþ independent uptake was measured in the presence of choline chloride, rather than KCl, as in the fragment studies. There are significant differences in the proportion of uptake by different components, primarily due to a difference in the Naþ-dependent uptake which was not mediated by system A. Significant differences between the MVM vesicles and villous fragments are indicated by *P < 0.05 vs villous fragments, fP < 0.001 vs fragments. Data is mean SEM.
mediates it? The reason for the apparent difference in the unidentified proportion of Naþ-dependent serine uptake component between vesicles and fragments is not clear although there are several possible explanations. The villous fragments contain other cell types which, although surrounded by syncytiotrophoblast, could contribute to uptake, particularly at the cut ends. In MVM vesicles transport proteins may be damaged during the purification process or lose an intracellular cofactor required for their function. Any transporters stored in vesicles within the cytoplasm would also be lost in the MVM vesicle preparations whereas these could be recruited to the MVM in villous fragments [12–14]. Other explanations may include differences in the Naþ gradient in the two systems or due to the gradient of another co-transported ion (e.g. Cl, Kþ or Hþ) which could selectively affect particular transport systems. Trans-stimulation by intracellular amino acids in the villous fragments might have been expected to cause a relative increase in System L activity in villous fragments, but this did not appear to be the case. As villous fragments are metabolically active, the oxidation of serine may have resulted in loss of tracer as CO2. While this could mean that the magnitude of serine uptake is underestimated, the proportions of uptake by different systems will remain the same. If
the unidentified Naþ-dependent component were temperature sensitive, this could also contribute to the observed difference between the two techniques. We attempted to address this issue by measuring serine uptake into MVM vesicles at 37 C. However, under these conditions serine uptake into MVM vesicles was not linear with time and we were not able to resolve the issue. Transporters known to mediate Naþ-dependent serine uptake in placental tissue include the System A isoforms SNAT1, 2 and 4 (SLC38A1, 2 and 4) and ASC (SCL1A4 and 5) [11]. Naþ-dependent serine transporters not known to be active in placenta include the System B0 isoform B0AT1 (SCL6A19), System B0,þ (SLC6A15) and the System N isoform SNAT5 (SLC38A5) based on functional studies in MVM and BM vesicles. However, the most likely candidates for this unidentified Naþ-dependent system, based on substrate specificity and known localisation to the syncytiotrophoblast, would be isoforms of System A and System ASC. The System A isoforms SNAT1 and 2 should be completely inhibited by the concentrations of MeAIB used in these studies. However it is possible that SNAT4, which has a lower affinity for MeAIB is not fully inhibited by the concentration of MeAIB used in the fragment studies [15]. Although SNAT4 activity is demonstrable
5
proportional activit y
Placental villous fragments MVM vesicles standard protocol MVM vesicles adapted protocol
4
3
2
φ φ
φ
φ
φ
φ
*
*
1
0 Na+ dependent / Na+ independent
System A / System L
Unknown Na+ dependent / Na+ independent
System A / Na+ dependent
System L / Na+ independent
Fig. 2. Relative serine uptake by different transport components in placental villous fragments and MVM vesicles measured in the same placentas. While the relative activity of system A and system L were not significantly different there were differences between the techniques in relation to Naþ dependent/Naþ independent activity and system A/Naþ dependent activity. This indicates that while there are differences between the methodologies for some transport components others, such as system A and system L, are comparable. *P < 0.05 vs villous fragments, fP < 0.001 vs villous fragments. Data is mean SEM, n ¼ 5 or 6.
A.P. Brand et al. / Placenta 31 (2010) 456–459
in first trimester placental villous fragments and MVM vesicles isolated from term placentas, there is little or no detectable SNAT4 activity in term placental villous fragments [16]. As such we think it is unlikely that this component of serine uptake is SNAT4mediated. System ASC activity has been demonstrated in the BM but not MVM of placental syncytiotrophoblast [17,18]. If the unidentified Naþ-dependent component of serine uptake was by System ASC, our study suggests it has low activity in vesicles and there may not have been enough activity for definitive characterisation. Without further experimentation it is not clear which transport system is mediating the unidentified Naþ-dependent serine uptake. While SNAT4 or System ASC would be the most obvious candidates, other transport systems cannot be ruled out. These techniques are important for the study of amino aid transporters in fetal growth restriction. Most work in this area has focused on systems A and L which were found to be comparable between the two techniques. However this work does raise the question as to how well these techniques represent less well described transport components, which might be important in fetal growth restriction. In conclusion, we have shown that key components of serine uptake are comparable between techniques but that for specific components of L-serine uptake, such as Systems A and L, there are significant differences between the MVM vesicle and placental villous fragment techniques. This provides reassurance that both models can be exploited to examine changes in placental transporter activity in well-characterised transporters. This notion is further supported by the similar trend observed between MVM vesicles and placental villous fragments with regard to the gestational increase in System A activity [16,19] and the reduced activity of Systems A [20,21] and L [22,23] associated with fetal growth restriction.
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17] [18] [19]
References [1] Glazier JD, Sibley CP. In vitro methods for studying human placental amino acid transport: placental plasma membrane vesicles. Methods Mol Med 2006;122:241–52. [2] Greenwood SL, Sibley CP. In vitro methods for studying human placental amino acid transport placental villous fragments. Methods Mol Med 2006;122:253–64. [3] Ayuk PT, Sibley CP, Donnai P, D’Souza S, Glazier JD. Development and polarization of cationic amino acid transporters and regulators in the human placenta. Am J Physiol Cell Physiol 2000;278:C1162–71. [4] Lewis RM, Glazier J, Greenwood SL, Bennett EJ, Godfrey KM, Jackson AA, et al. L-serine uptake by human placental microvillous membrane vesicles. Placenta 2007;28:445–52. [5] Godfrey KM, Matthews N, Glazier J, Jackson A, Wilman C, Sibley CP. Neutral amino acid uptake by the microvillous plasma membrane of the human
[20]
[21]
[22] [23]
459
placenta is inversely related to fetal size at birth in normal pregnancy. J Clin Endocrinol Metab 1998;83:3320–6. Johansson M, Karlsson L, Wennergren M, Jansson T, Powell TL. Activity and protein expression of Naþ/Kþ ATPase are reduced in microvillous syncytiotrophoblast plasma membranes isolated from pregnancies complicated by intrauterine growth restriction. J Clin Endocrinol Metab 2003;88:2831–7. Magnusson AL, Waterman IJ, Wennergren M, Jansson T, Powell TL. Triglyceride hydrolase activities and expression of fatty acid binding proteins in the human placenta in pregnancies complicated by intrauterine growth restriction and diabetes. J Clin Endocrinol Metab 2004;89:4607–14. Glazier JD, Jones CJ, Sibley CP. Purification and Naþ uptake by human placental microvillus membrane vesicles prepared by three different methods. Biochim Biophys Acta 1988;945:127–34. Illsley NP, Wang ZQ, Gray A, Sellers MC, Jacobs MM. Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta 1990;1029:218–26. Jimenez V, Henriquez M, Llanos P, Riquelme G. Isolation and purification of human placental plasma membranes from normal and pre-eclamptic pregnancies. A comparative study. Placenta 2004;25:422–37. Cleal JK, Lewis RM. The mechanisms and regulation of placental amino acid transport to the human foetus. J Neuroendocrinol 2008;20:419–26. Ling R, Bridges CC, Sugawara M, Leibach FH, Prasad PD, Ganapathy V, et al. Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A. Biochim Biophys Acta 2001;1512:15–21. Hatanaka T, Hatanaka Y, Tsuchida J, Ganapathy V, Setou M. Amino acid transporter ATA2 is stored at the trans-golgi network and released by insulin stimulus in adipocytes. J Biol Chem 2006;281:39273–84. Parrott MS, von Versen-Hoeynck F, Ness RB, Markovic N, Roberts JM. System A amino acid transporter activity in term placenta is substrate specific and inversely related to amino acid concentration. Reprod Sci 2007;14:687–93. Hatanaka T, Huang W, Ling R, Prasad PD, Sugawara M, Leibach FH, et al. Evidence for the transport of neutral as well as cationic amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A. Biochim Biophys Acta 2001;1510:10–7. Desforges M, Mynett KJ, Jones RL, Greenwood SL, Westwood M, Sibley CP, et al. The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane. J Physiol 2009;587:61–72. Hoeltzli SD, Smith CH. Alanine transport systems in isolated basal plasma membrane of human placenta. Am J Physiol 1989;256:C630–7. Johnson LW, Smith CH. Neutral amino acid transport systems of microvillous membrane of human placenta. Am J Physiol 1988;254:C773–80. Mahendran D, Byrne S, Donnai P, D’Souza SW, Glazier JD, Jones CJ, et al. Naþ transport, Hþ concentration gradient dissipation, and system A amino acid transporter activity in purified microvillous plasma membrane isolated from first-trimester human placenta: comparison with the term microvillous membrane. Am J Obstet Gynecol 1994;171:1534–40. Mahendran D, Donnai P, Glazier JD, D’Souza SW, Boyd RD, Sibley CP. Amino acid (system A) transporter activity in microvillous membrane vesicles from the placentas of appropriate and small for gestational age babies. Pediatr Res 1993;34:661–5. Shibata E, Hubel CA, Powers RW, von Versen-Hoeynck F, Gammill H, Rajakumar A, et al. Placental system A amino acid transport is reduced in pregnancies with small for gestational age (SGA) infants but not in preeclampsia with SGA infants. Placenta 2008;29:879–82. Jansson T, Scholtbach V, Powell TL. Placental transport of leucine and lysine is reduced in intrauterine growth restriction. Pediatr Res 1998;44:532–7. Roos S, Jansson N, Palmberg I, Saljo K, Powell TL, Jansson T. Mammalian target of rapamycin in the human placenta regulates leucine transport and is downregulated in restricted fetal growth. J Physiol 2007;582:449–59.